Cell Cycle Modulation of Gene Targeting by a Triple Helix-forming Oligonucleotide

Abstract

Successful gene-targeting reagents must be functional under physiological conditions and must bind chromosomal target sequences embedded in chromatin. Triple helix-forming oligonucleotides (TFOs) recognize and bind specific sequences via the major groove of duplex DNA and may have potential for gene targeting in vivo. We have constructed chemically modified, psoralenlinked TFOs that mediate site-specific mutagenesis of a chromosomal gene in living cells. Here we show that targeting efficiency is sensitive to the biology of the cell, specifically, cell cycle status. Targeted mutagenesis was variable across the cycle with the greatest activity in S phase. This was the result of differential TFO binding as measured by cross-link formation. Targeted cross-linking was low in quiescent cells but substantially enhanced in S phase cells with adducts in ϳ20 -30% of target sequences. 75-80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could be recovered by direct screening of colonies chosen at random. These results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFOmediated manipulation of chromosomal sequences are likely to reflect a combination of appropriate oligonucleotide chemistry and manipulation of the cell biology.Effective gene-targeting reagents would have a broad application as probes of chromatin structure and in a variety of genomic manipulations, e.g. gene knock-out, targeted gene conversion and/or recombination, and gene therapy. Successful constructs must be functional in the nuclear environment of living cells. Furthermore, their intended targets must be accessible despite the packaging and condensation of nuclear DNA by chromosomal proteins. One targeting strategy that has been of interest for many years is based on triple-helix forming oligonucleotides (TFOs). 1A DNA triple helix can form when a TFO lies in the major groove of intact duplex DNA (1-5). The most stable structures assemble on polypurine:polypyrimidine sequences with hydrogen bonds formed between the bases in the third strand and those in the purine strand of the duplex. The purine or pyrimidine motif third strands may be involved in triplex formation depending on the target sequence, and a binding code for the design of the third strands has been defined (6). Although conventional oligonucleotides have features that limit their activity under physiological conditions, there is a variety of chemical modifications that ameliorate these limitations (7). Thus, for pyrimidine TFOs, the replacement of cytosine by 5-methylcytosine and the use of 2Ј-O-methyl (2Ј-OMe)-modified sugars permit stable triplex formation at physiological pH in vitro (8 -13). Other derivatives contribute to the bioactivity of TFOs of both motifs as shown by us and others in recent publications (14 -18).Although chemical modifications can enhance TFO affinity and triplex stability, they obviously cannot add...

Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

“…To further explore the cell-cycle dependence of gene correction induced by bis-PNAs in the CHO-GFP/IVS2-1 G3A reporter cells, we investigated bis-PNA activity in other stages of the cell cycle. We synchronized cells in various cell cycle stages using established CHO cell synchronization protocols (18), and transfected cells in each phase by electroporation, followed by chloroquine treatment. As shown in Fig.…”

Gene therapy progress and prospects: targeted gene repair

“…11,36,41,42 In a passive role, one could envision that DNA replication functions simply to destabilize chromatin around the target site making it more accessible to the oligo. However, replication could play a more direct and dynamic role in the mechanism of correction.…”

“…DNA lesions induced by drugs such as VP16 or CPT activate HR and retard, stall or inhibit replication. [10][11][12]41,42,46 All these events elevate gene repair activity but are also subject to downregulation or even suppression by p53 or MSH2. While the evidence is sketchy, it is plausible that DNA damage other than simple mispairs could activate the suppressive MMR Targeted gene repair: progress and prospects H Parekh-Olmedo et al activity.…”

“…This model would also predict that cells in S phase are more amenable to gene repair activity and recent data strongly suggest that this is the case. 24,36,42,48 Correction-yes, but at what price?…”

Chemical Tools for Targeted Mutagenesis of DNA Based on Triple Helix Formation

“…These mutagenesis experiments show that the packaging of the DNA into chromatin is not an absolute barrier to gene targeting with TFOs. 43) Successful procedures for the gene manipulation of chromosomal sequences using TFOs will require their high affinity to the target genes under physiologic conditions.…”